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Abstract:

A process for preparing a nicotine-based hapten of formula I
##STR00001##
or a salt or solvate thereof, wherein n, R1 and R2 have the
meanings as indicated in the specification. The nicotine-based hapten
forms part of a smoking cessation vaccine.

Claims:

1. A process for preparing a nicotine-based hapten of formula I
##STR00023## or a salt or solvate thereof, wherein n is an integer from
0 to 5, and R1 and R2 together form a N-bonded 5- to
10-membered heterocyclic group containing from 1 to 4 ring nitrogen atoms
and optionally containing from 1 to 4 other heteroatoms selected from the
group consisting of oxygen and sulfur, said heterocyclic group being
optionally substituted at 1, 2, 3 or 4 positions by halo, cyano, hydroxy,
oxo, amino, aminocarbonyl, nitro, C1-C4-alkyl,
C1-C4-alkoxy or C3-C6-cycloalkyl, the process
comprising the steps of: (a) reacting a compound of formula II
##STR00024## or a salt or solvate thereof, wherein n is an integer from
0 to 5, with a compound of formula III ##STR00025## or a salt thereof,
wherein R1 and R2 together form a N-bonded 5- to 10-membered
heterocyclic group containing from 1 to 4 ring nitrogen atoms and
optionally containing from 1 to 4 other heteroatoms selected from the
group consisting of oxygen and sulfur, said heterocyclic group being
optionally substituted at 1, 2, 3 or 4 positions by halo, cyano, hydroxy,
oxo, amino, aminocarbonyl, nitro, C1-C4-alkyl,
C1-C4-alkoxy or C3-C6-cycloalkyl, and a
polymer-supported coupling agent; and (b) filtering the product of step
(a) to give a compound of formula I in free, salt or solvate form,
wherein n is an integer from 0 to 5, and R1 and R2 together
form a N-bonded 5- to 10-membered heterocyclic group containing from 1 to
4 ring nitrogen atoms and optionally containing from 1 to 4 other
heteroatoms selected from the group consisting of oxygen and sulfur, said
heterocyclic group being optionally substituted at 1, 2, 3 or 4 positions
by halo, cyano, hydroxy, oxo, amino, aminocarbonyl, nitro,
C1-C4-alkyl, C1-C4-alkoxy or
C3-C6-cycloalkyl.

2. A process according to claim 1, wherein n of the compound of formula
II is 1.

3. A process according to claim 1, wherein R1 and R2 of the
compound of formula III together form a N-bonded 5- to 6-membered
heterocyclic group containing from 1 or 2 ring nitrogen atoms and
optionally containing 1 or 2 other heteroatoms selected from the group
consisting of oxygen and sulfur, said heterocyclic group being optionally
substituted at 1 or 2 positions by halo, cyano, hydroxy, oxo, amino,
aminocarbonyl, nitro, C1-C4-alkyl, C1-C4-alkoxy or
C3-C6-cycloalkyl.

4. A process according to claim 3, wherein R1 and R2 of the
compound of formula III together form a pyrrolidine group substituted at
positions 2 and 5 by oxo.

5. A process according to claim 1, wherein the polymer-supported coupling
agent is a compound of formula IV ##STR00026## wherein W denotes a
solid phase substrate chemically linked to the indicated methylene group
and R3 is C1-C5-alkyl or C3-C8-cycloalkyl.

6. A process according to claim 5, wherein R3 of the compound of
formula IV is ethyl, isopropyl or cyclohexyl.

7. A process according to claim 1, wherein step (a) is carried out at a
temperature from 40.degree. C. to 60.degree. C.

8. A process according to claim 1, wherein step (a) is carried out in
2-butanone as solvent.

9. A process for preparing a nicotine-based hapten of formula I as
defined in claim 1 that is also a compound of formula Ib ##STR00027##
or a salt or solvate thereof, the process comprising the steps of: (a)
reacting a compound of formula IIa ##STR00028## or a salt or solvate
thereof, with N-hydroxy succinimide or a salt thereof, and a
polymer-supported coupling agent of formula IV ##STR00029## wherein W
denotes a solid phase substrate chemically linked to the indicated
methylene group and R3 is C1-C5-alkyl or
C3-C8-cycloalkyl; and (b) filtering the product of step (b) to
give a compound of formula Ib in free, salt or solvate form.

10. A method for preparing a nicotine-based hapten-carrier conjugate, the
method comprising covalently coupling a nicotine-based hapten of formula
I obtainable by the process of claim 1 to one or more coat proteins of a
virus-like particle (VLP).

11. A method according to claim 10 wherein the VLP comprises coat
proteins of an RNA phage.

Description:

FIELD OF THE INVENTION

[0001] This invention relates to a process for preparing nicotine-based
haptens that are useful components of smoking cessation vaccines.

BACKGROUND TO THE INVENTION

[0002] Tobacco is the world's most widely used addictive drug. The
principal addictive component of tobacco is nicotine, an alkaloid derived
from tobacco leaves. The smoking of tobacco in cigarettes is the single
leading cause of preventable death in the United States and many other
countries. While most smokers are aware that lung cancer, coronary heart
disease, chronic lung disease and stroke can be caused by smoking, the
majority of cigarette smokers who try to quit fail to do so.

[0003] Various behavioural and pharmacologic treatments are available to
help smokers quit including nicotine replacement therapy (e.g. using
nicotine gums or transdermal patches) and antidepressant treatment (e.g.
using bupropion). However results are decidedly mixed. They can give rise
to undesirable side effects. And they rely heavily on smokers maintaining
their will power not to smoke.

[0004] U.S. Pat. No. 6,932,971 discloses a therapeutic vaccine for smoking
cessation. When administered the vaccine induces the production of the
high levels of nicotine-specific antibodies that bind nicotine in the
blood. As the complex of nicotine attached to an antibody is too large to
pass the blood-brain-barrier, nicotine uptake into the brain and the
subsequent stimulation of nicotine-perceptive neurons in the brain is
believed to be significantly reduced or even prevented. In this way the
addiction-driving and satisfaction-inducing stimulus of nicotine is
minimized.

[0005] The vaccine of U.S. Pat. No. 6,932,971 consists of a nicotine-based
hapten linked via a chemical bridge to the surface of a phagus Qβ
recombinant virus-like-particle produced in E. coli. The nicotine-based
hapten is a known nicotine derivative,
1-(trans-3'-hydroxymethyl-nicotinyl)-6-hydroxy-succinimidyl-succinate
(C19H23N3O6). This hapten is synthesized by reacting
trans-3'-hydroxymethylnicotine with succinic anhydride to yield the
succinylated hydroxymethyl-nicotine,
O-succinyl-3'-hydroxymethyl-nicotine, based on the procedure disclosed in
Langone et al "Radioimmuno-assay of nicotine, cotine and
γ-(3-pyridyl)-γ-oxo-N-methylbutyramide", Methods Enzymol. 84,
pages 628-640, Academic Press 1982. This compound is then mixed with
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and
N-hydroxysuccinimide (NHS) to give the N-hydroxysuccinimide ester of
O-succinyl-3'-hydroxymethyl-nicotine, namely the aforementioned
1-(trans-3'-hydroxymethyl-nicotinyl)-6-hydroxysuccinimidyl-succinate.

[0006] The process disclosed in U.S. Pat. No. 6,932,971 is undesirable for
industrial production as the hapten that is prepared by the process can
contain residual amounts of EDC, which is toxic and mutagenic. The hapten
is also an inherently unstable molecule. This makes handling and
preparation of the hapten difficult. There is therefore a need for a
process for preparing the hapten that avoids or at least minimizes these
problems.

SUMMARY OF THE INVENTION

[0007] In a first aspect, the present invention relates to a process for
preparing a nicotine-based hapten of formula I

##STR00002##

or a salt or solvate thereof

[0008] wherein n is an integer from 0 to 5, and R1 and R2
together form a N-bonded 5- to 10-membered heterocyclic group containing
from 1 to 4 ring nitrogen atoms and optionally containing from 1 to 4
other heteroatoms selected from the group consisting of oxygen and
sulfur, said heterocyclic group being optionally substituted at 1, 2, 3
or 4 positions by halo, cyano, hydroxy, oxo, amino, aminocarbonyl, nitro,
C1-C4-alkyl, C1-C4-alkoxy or
C3-C6-cycloalkyl; the process comprising the steps of:

[0009] (a) reacting a compound of formula II

##STR00003## [0010] or a salt or solvate thereof, wherein n is an
integer from 0 to 5,

[0011] with a compound of formula III

##STR00004## [0012] or a salt thereof, [0013] wherein R1 and
R2 are as hereinbefore defined, [0014] and a polymer-supported
coupling agent; and [0015] (b) filtering the product of step (a) to give
a compound of formula I in free, salt or solvate form, as hereinbefore
defined.

[0016] In a second aspect the present invention relates to a
nicotine-based hapten of formula I as hereinbefore defined obtainable or
obtained by the aforementioned process for preparing a nicotine-based
hapten of formula I. Preferably, the hapten is provided as a composition
wherein the hapten is greater than 80, 85 or 90% pure, as determined, for
example, by 1H NMR.

[0017] In a third aspect the present invention provides a method for
preparing a hapten-carrier conjugate, the method comprising covalently
coupling a nicotine-based hapten of formula I obtained or obtainable by
the process of the first aspect of the invention to one or more coat
proteins of a virus like particle (VLP). In one embodiment, the VLP
comprises coat proteins of an RNA phage, preferably phage Qβ.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Terms used in the specification have the following meanings:

[0019] "Optionally substituted" as used herein means the group referred to
can be substituted at one or more positions by any one or any combination
of the radicals listed thereafter.

[0020] "Halo" or "halogen" as used herein may be fluorine, chlorine,
bromine or iodine. Halo is suitably chlorine.

[0021] "C1-C5-alkyl" as used herein denotes straight chain or
branched alkyl having 1 to 5 carbon atoms. C1-C5-alkyl is
suitably methyl or ethyl.

[0022] "C3-C8-cycloalkyl" as used herein denotes cycloalkyl
having 3 to 8 ring carbon atoms, for example a monocyclic group such as a
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or
cyclooctyl, or a bicyclic group such as bicycloheptyl or bicyclooctyl.
C3-C8-cycloalkyl" is suitably C5-C6-cycloalkyl,
especially cyclohexyl.

[0023] "N-bonded 5- to 10-membered heterocyclic group containing from 1 to
4 ring nitrogen atoms and optionally containing from 1 to 4 other
heteroatoms selected from the group consisting of oxygen and sulfur" as
used herein is a heterocyclic group that contains 5, 6, 7, 8, 9, or 10
ring atoms, one of which is nitrogen and is attached to the oxygen atom
of the ester group that is distal to the nicotinyl moiety of the compound
of formula I, wherein optionally 1, 2 or 3 of the other ring atoms are
nitrogen atoms and optionally 1, 2, 3 or 4 of the other ring atoms can be
selected from oxygen and sulfur. The N-bonded 5- to 10-membered
heterocyclic group may be, for example a saturated or an unsaturated,
mono-cyclic or bicyclic heterocyclic group. The N-bonded 5- to
10-membered heterocyclic group is suitably a N-bonded 5- or 6-membered
heterocyclic group containing from 1 to 4 ring nitrogen atoms, especially
N-bonded pyrrolidinyl.

[0024] "Nicotine-based hapten" as used herein refers to nicotine, either
in its enantiomerically pure S or R form or a mixture thereof, which is
derivatised in such manner so as to attachable to a carrier either
directly, or via a cross-linker.

[0025] "Vaccine" as used herein refers to a formulation which contains the
nicotine-based hapten of the present invention linked to a carrier and
which is in a form that is capable of being administered to an animal.
Typically, the vaccine comprises a conventional saline or buffered
aqueous solution medium, for example an aluminium salt solution, in which
the nicotine-based hapten-carrier conjugate is suspended or dissolved. In
this form, the vaccine can be used-conveniently to prevent, ameliorate,
or otherwise treat a condition. Upon introduction into a host, the
vaccine is able to provoke an immune response including, but not limited
to, the production of antibodies and/or cytokines and/or the activation
of cytotoxic T cells, antigen presenting cells (e.g. immunoglobulins),
helper T cells, dendritic cells and/or other cellular responses.

[0026] Throughout this specification and in the claims that follow, unless
the context requires otherwise, the word "comprise", or variations such
as "comprises" or "comprising", will be understood to imply the inclusion
of a stated integer or step or group of integers or steps but not the
exclusion of any other integer or step or group of integers or steps.

[0027] The present invention relates to a process for preparing
nicotine-based haptens that are useful components of smoking cessation
vaccines.

[0028] Smoking cessation vaccines that contain nicotine-based haptens are
disclosed in U.S. Pat. No. 6,932,971 (Cytos), U.S. Pat. No. 6,232,082
(Nabi) and U.S. Pat. No. 6,656,469 (IPAB) where the haptens are linked
via a chemical bridge to a phagus Qβ recombinant virus-like-particle
produced in E. coli., recombinant Psuedomonas aeruginosa exoprotein A
(rEPA) and a tetanus toxoid respectively.

[0029] Nicotine has the following chemical structure:

##STR00005##

[0030] Nicotine does not provoke an immunological response in man. However
it is possible to generate nicotine-specific antibodies in man when
nicotine is derivatised to form a hapten that is linked to a suitable
carrier.

[0031] The present invention relates to a process for preparing
nicotine-based haptens of formula I:

##STR00006##

in free, salt or solvate form, wherein n is an integer from 0 to 5, and
R1 and R2 together form a N-bonded 5- to 10-membered
heterocyclic group containing from 1 to 4 ring nitrogen atoms and
optionally containing from 1 to 4 other heteroatoms selected from the
group consisting of oxygen and sulfur, said heterocyclic group being
optionally substituted at 1, 2, 3 or 4 positions by halo, cyano, hydroxy,
oxo, amino, aminocarbonyl, nitro, C1-C4-alkyl,
C1-C4-alkoxy or C3-C6-cycloalkyl.

[0032] The following suitable, preferred, more preferred or most preferred
aspects of the invention may be incorporated independently, collectively
or in any combination.

[0033] n is suitably an integer from 0 to 5, for example 0, 1, 2, 3, 4 or
5, but especially 1.

[0034] R1 and R2 together suitably form a N-bonded 5- to
6-membered heterocyclic group containing from 1 or 2 ring nitrogen atoms
and optionally containing 1 or 2 other heteroatoms selected from the
group consisting of oxygen and sulfur, said heterocyclic group being
optionally substituted at 1 or 2 positions by halo, cyano, hydroxy, oxo,
amino, aminocarbonyl, nitro, C1-C4-alkyl,
C1-C4-alkoxy or C3-C6-cycloalkyl, especially oxo. For
example, R1 and R2 together a pyrrolidine group substituted at
positions 2 and 5 by oxo i.e. succinimidyl.

[0035] When the aforementioned N-bonded 5- to 6-membered heterocyclic
group is substituted by C1-C4-alkyl, C1-C4-alkyl is
suitably methyl or ethyl.

[0036] When the aforementioned N-bonded 5- to 6-membered heterocyclic
group is substituted by C1-C4-alkoxy, C1-C4-alkoxy is
suitably methoxy or ethoxy.

[0037] When the aforementioned N-bonded 5- to 6-membered heterocyclic
group is substituted by C3-C6-cycloalkyl,
C3-C6-cycloalkyl is suitably pentyl or hexyl.

[0038] The haptens include at least one asymmetric carbon atom so they
exist in individual isomeric forms or as mixtures thereof, e.g. as
racemic or diastereomeric mixtures. The present invention embraces
preparing all individual isomers of each stereocentre (e.g. SS SR RS and
RR isomers) as well as mixtures, e.g. racemic (e.g. 50:50 of two isomers
or 25:25:25:25 of all four isomers); or diastereomeric mixtures, thereof.

[0039] Nicotine-based haptens of formula I are suitably nicotine-based
haptens of formula Ia

##STR00007##

in free, salt or solvate form, which is a racemic mixture of trans
enantiomers.

[0040] In an especially preferred embodiment the nicotine-based hapten of
formula I is a nicotine-based hapten of formula Ib

##STR00008##

in free, salt or solvate form, which is a racemate of trans-4-nicotine
methylene mono-succinate ester succinimidyl ester, also known as trans
succinic acid 2,5-dioxo-pyrrolidin-1-yl ester
1-methyl-2-pyridin-3-yl-pyrrolidin-3-ylmethyl ester
(C19H23N3O6).

[0041] The process for preparing the nicotine-based haptens of formula I
comprises two steps, (a) and (b), which can be performed in a standard
reactor (preferably with slow stirring in order not to damage the polymer
structure) and filtration in a standard unit.

[0042] In step (a) a compound of formula II

##STR00009##

or a salt or solvate thereof,

[0043] wherein n is an integer from 0 to 5, is reacted with a compound of
formula III

##STR00010##

or a salt thereof,

[0044] wherein R1 and R2 are as hereinbefore defined,

[0045] and with a polymer-supported coupling agent.

[0046] In a preferred embodiment the compound of formula II is a compound
of formula IIa

##STR00011##

in free, salt or solvate form.

[0047] The polymer-supported coupling agent is suitably a
polymer-supported solution phase synthesis reagent for producing
activated acid species in the formation of amide bonds and esters.

[0048] In a preferred embodiment the polymer-supported coupling agent is a
compound of formula IV

##STR00012##

wherein W denotes a solid phase substrate chemically linked to the
indicated methylene group and R3 is C1-C5-alkyl or
C3-C8-cycloalkyl.

[0049] When R3 is C1-C5-alkyl, it is suitably ethyl or
isopropyl.

[0050] When R3 is C3-C8-cycloalkyl, it is suitably
cyclohexyl.

[0051] In an especially preferred embodiment the compound of formula IV is
suitably a cyclohexyl carbodiimide resin that is commercially available
from Varian Inc as StratoSpheres® PL-DCC resin.

[0052] An advantage of using a polymer-supported coupling agent is that
toxic by-products remain bound to the polymeric support thereby greatly
simplifying the work-up procedure by avoiding the need for aqueous
extractions and the removal of the solid by-product (the
dicyclohexylurea). Any unreacted acid or amine species can be removed by
adding an appropriate scavenger resin.

[0053] The compounds of formula II and III are preferably mixed before
coming into contact with the polymer-supported coupling agent. It should
be noted mixing the compound of formula II with the polymer-supported
coupling agent before admixing the compound of formula III can diminish
yield of nicotine-based haptens of formula I and even lead to alternative
products being formed.

[0054] The reaction may be effected using known methods for reacting
carboxylic acids with amino compounds and polymer-supported coupling
agent (e.g. substrate-bound carbodiimide derivatives), or analogously
e.g. as hereinafter described in the Examples. The reaction is
conveniently carried out using an organic solvent such as 2-butanone
(also known as ethyl methyl ketone or butan-2-one) i.e. the compounds of
formula II and III are dissolved in the solvent in a first vessel and the
polymer-supported coupling agent is swelled with the same solvent in a
second vessel. The contents of the first and second vessels are combined
so that the compounds of formula II and III react with an intermediate of
the polymer-supported coupling agent. Suitable reaction temperatures are
from 20° C. to 70° C., preferably from 40° C. to
60° C., but especially about from 50° C.

[0055] Increasing the temperature of the reaction would most likely
increase the reaction rate, but this tends to increase the quantity and
number of side-products. Higher temperatures can lead to nucleophilic
ring opening of the succinimidyl group followed by a rearrangement.

[0056] In step (b) the product of step (a) is filtered to give the
nicotine-based hapten of formula I in solution.

[0057] Filtering can be achieved by any art-known means.

[0058] The final product is of high purity, especially when compared to
the laboratory scale process described in U.S. Pat. No. 6,932,971. This
is particularly important when preparing the hapten for clinically
testing the vaccine. It is often difficult to purify nicotine-based
haptens as this tends to lead to degradation through hydrolysis.
Preferably, the purity of the nicotine hapten is greater than 80, 85 or
90% pure, as determined, for example, by 1H NMR.

[0059] If desired the resulting solid form is dissolved in a suitable
organic solvent, for example 2-butanone, for shipping and storage, which
is suitably at a reduced temperature i.e. from -100° C. to
20° C., for example about -80° C.

[0060] Compounds of formula II are commercially available or may be
prepared by reacting a compound of formula V

##STR00013##

[0061] with a compound of formula VI

##STR00014##

wherein n is an integer from 0 to 5. The reaction may be effected using
known methods for reacting dihydro-furan-2,5-dione or analogues with
alcohols, or analogously e.g. as hereinafter described in the Examples.

[0062] Compounds of formula III are either commercially available or may
be obtained by known procedures for preparing hydroxylated N-heterocyclic
compounds.

[0063] Compounds of formula IV are commercially available.

[0064] The compound of formula V may be obtained by reducing a compound of
formula VII,

##STR00015##

namely, 1-methyl-5-oxo-2-pyridin-3-yl-pyrrolidine-3-carboxylic acid
methyl ester. The reaction may be effected using known methods for
reducing esters, preferably using a reducing agent such as lithium
aluminium hydride, or analogously e.g. as hereinafter described in the
Examples.

[0065] Compounds of formula VI are commercially available.

[0066] The compound of formula VII may be obtained by esterifying a
compound of formula VIII,

##STR00016##

namely, 1-methyl-5-oxo-2-pyridin-3-yl-pyrrolidine-3-carboxylic acid. The
reaction may be effected using known methods for esterifying carboxylic
acids with alcohols to form esters, preferably using a dehydrating agent
such as thionyl chloride (SOCl2), or analogously e.g. as hereinafter
described in the Examples.

[0067] in a preferred embodiment the present invention is a process for
preparing a nicotine-based hapten of formula Ib

##STR00017##

or a salt or solvate thereof, the process comprising the steps of:

[0068] (a) reacting a compound of formula IIa

##STR00018##

[0069] with N-hydroxy succinimide and a polymer-supported coupling agent
of formula IV

##STR00019## [0070] wherein W denotes a solid phase substrate
chemically linked to the indicated methylene group and R3 is
C1-C5-alkyl or C3-C8-cycloalkyl; and

[0071] (b) filtering the product of step (a) to give a compound of formula
Ib in free, salt or solvate form.

[0072] Haptens prepared by the process of the present invention can be
prepared as the free-base, or a salt or solvate form. If desired or
necessary, they may be converted into various salt forms or solvates
(suitably using a non-alcoholic solvent), and vice versa, in a
conventional manner. Isomers, such as enantiomers and diastereomers, may
be obtained in a conventional manner, e.g. by asymmetric synthesis from
correspondingly asymmetrically substituted, e.g. optically active,
starting materials or optically active asymmetric catalysts or optically
active auxiliaries.

[0073] Haptens prepared by the process of the present invention are useful
components of smoking cessation vaccines. To make such vaccines the
hapten is covalently conjugated to suitable carrier, e.g. a virus-like
particle (VLP), such as a VLP based on the coat proteins of an RNA phage,
preferably RNA phage Qβ, as described in WO04/009116. Suitable
processes are described in WO04/009116, for example a process where the
derivatized nicotine hapten reacts with lysine residues present on the
surface of the virus-like particle coat proteins to form an amide bond.
The hapten-carrier conjugate is then combined with one or more
pharmaceutically acceptable formulation ingredients. Suitable
formulations are, for example, described in WO2007/131972, where the
formulation includes at least one non-reducing saccharide, e.g. sucrose
or trehalose, and at least one non-ionic surfactant, preferably to give a
pH of from 5.4 to 6.6. In one embodiment, such formulations are
lyophilized.

[0074] In one embodiment of the present invention, the composition further
comprises an adjuvant, which preferably is aluminum containing adjuvant,
preferably an aluminum salt, preferably aluminium hydroxide, preferably
an aluminum containing mineral gel, most preferably alhydrogel. In one
preferred embodiment of the present invention, the composition comprises
from 1 mg to 2 mg, preferably from 1.2 mg to 1.7 mg, more preferably from
1.3 mg to 1.5 mg of aluminium salt, preferably aluminium hydroxide.

[0075] The vaccine is typically injected into human patients desiring an
aid to smoking cessation. Suitable dosages and dosage regiments are
described in WO2008/129020. For example, the dosage regiment may comprise
at least a first, a second and a third administration into the human of
the composition, wherein the time interval between the first
administration and the second administration, and between the second
administration and the third administration is at most 18 days.
Preferably the time interval between the first administration and the
second administration, and between the second administration and the
third administration is at least three days, preferably at least four
days, more preferably at least five days. In one preferred embodiment,
the time interval between the first administration and the second
administration, and between the second administration and the third
administration is at least five days and at most 18 days.

[0076] In one preferred embodiment, during each administration a dose of
at least 50 μg of the hapten-carrier conjugate, preferably at least
100 μg, or preferably at least 200 μg or at least 300 μg is
administered. The dose of the hapten-carrier conjugate preferably shall
not exceed 500 μg, preferably not exceeding 400 μg. In one
preferred embodiment of the present invention, during each administration
about 100 μg of the hapten-carrier conjugate is administered. The
compositions may be administered by various methods known in the art, but
will normally be administered by injection, infusion, inhalation, oral
administration, or other suitable physical methods. The compositions may
alternatively be administered intramuscularly, intravenously,
transmucosally, transdermally or subcutaneously. In one very preferred
embodiment, the administration of the composition is administered
subcutaneously. Components of compositions for administration include
sterile aqueous (e.g., physiological saline) or non-aqueous solutions and
suspensions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and injectable
organic esters such as ethyl oleate

[0077] Antibodies against nicotine are induced by an immune reaction of
the body to the hapten-carrier conjugate particles. It is believed that
these antibodies will bind any inhaled nicotine preventing them passing
through the blood-brain barrier. As such, no nicotine binding to target
sites in the brain and no resulting dopamine release will occur which
provides the sensation of pleasure/reward. The patient will thereby be
more likely to continue to refrain from smoking.

[0078] The invention is described further by reference to the following
Examples, which are illustrative only and non-limiting.

EXAMPLES

Preparation of Starting Materials

Preparation of racemic trans-4-nicotine methylene alcohol

[0079] Racemic trans-4-cotinine carboxylic acid (1.8 kg) is added to
methanol (17 L) at room temperature, and the suspension heated to
35° C. (internal temperature)(Buechi glass-lined 50 L vessel).
Thionyl chloride (1.07 kg) is then added carefully within one hour at
35° C. The solution is left to stir for a further 1 hour upon
which an In-Process Control (HPLC conversion) is conducted to ensure
complete reaction. Toluene (12 L) is added to the reaction mixture, prior
to cooling to -5° C. (internal temperature) and the solution
carefully quenched with aqueous sodium hydroxide solution (2.5 kg, 10 N).
Filtration to remove NaCl is undertaken before washing of the cake with
methanol (6.3 L) and subsequent azeotropic removal of methanol and water
by repeat distillations from toluene (total volume toluene 48 L). A
second filtration (further NaCl removal) gives the racemic trans-methyl
trans-4-cotinine ester as a toluene solution (concentration in the range
of 9 m/m %). The solution is to be stored for a limited period due to
spontaneous crystallisation. The crystals can be re-dissolved on warming
to 50° C. (external temperature) for 1 hour.

[0080] The racemic trans-methyl-4-cotinine ester solution (1.91 kg as a
solution in toluene) is added slowly at room temperature to a lithium
aluminium hydride solution in THF (7.79 kg, 4.5 m/m %, Chemetall)(Buechi
glass-lined 100 L vessel). The reaction mixture is left to stir for 4
hours upon which cellflock (1.5 kg) is added and the reaction quenched
carefully with water (0.5 kg) After two hours of further stirring, the
insoluble lithium and aluminium salts are removed by filtration, and the
cake is washed with THF (13 L). The racemic trans-4-nicotine methylene
alcohol is obtained as a solution in THF/toluene (concentration in the
range of 4 m/m %).

[0081] Racemic trans-4-nicotine methylene alcohol as a solution in THF I
toluene (1.27 kg, concentration in the range of 4 m/m %) is heated to
55° C. (internal temperature) and distillation, followed by
acetonitrile addition (total volume 21.6 L) is repeated to provide the
alcohol in acetonitrile (Buechi glass-lined 50 L vessel). The solution of
the racemic trans-4-nicotine methylene alcohol (1.27 kg, as a solution in
acetonitrile in the range of 12 m/m %) is then heated to 70° C.
(internal temperature) to which a solution of succinic anhydride (VI) in
acetonitrile (668 g, 23 m/m %) is slowly added. The reaction mixture is
left to stir at 70° C. for 8 hours until sufficient conversion to
the product (II) is confirmed by In-Process Control (HPLC conversion). A
solvent switch to 2-butanone is made and the crude material
chromatographed over silica (15 kg) (Filtration plate apparatus, 20 L),
eluting the product with a 2-butanone/methanol mixture (1:1, 192 kg).
Following analysis for purity (HPLC), selected fractions are then
distilled and the solvent switched back to 100% 2-butanone.

[0084] Following successful In-Process Control (HPLC conversion), the
suspension is filtered to remove the polymeric reagent and by-products,
and the cake washed. The 2-butanone is then fully or partially removed by
distillation using a rotary evaporator. Racemic trans-4-nicotine
methylene mono-succinate ester succinimidyl ester is formed as a golden
oil when fully dried with a mass of 673 g.

[0085] The final product is determined to have a purity of >70% by HPLC
assay (area comparison to an aminated analogue) and ≧90% by
1H NMR.

Patent applications in all subclasses VIRUS OR BACTERIOPHAGE, EXCEPT FOR VIRAL VECTOR OR BACTERIOPHAGE VECTOR; COMPOSITION THEREOF; PREPARATION OR PURIFICATION THEREOF; PRODUCTION OF VIRAL SUBUNITS; MEDIA FOR PROPAGATING